Influenza viruses cause highly contagious, acute respiratory illnesses. In an average year influenza is associated with approximately 36,000 deaths and approximately $4.6 billion direct medical costs in the United States a much greater loss of life and economic prosperity has occurred during influenza pandemics. Because influenza virus polymerase possesses several virus-specific activities, small molecule drugs targeting the polymerase are likely to offer convenient, broad, and low-cost protection against influenza. Influenza virus polymerase consists of three proteins, PA, PB1 and PB2, and it has an unusual endonuclease activity. Transcription by the polymerase is initiated using a capped primer seized from host cellular mRNAs. In contrast, RNA replication does not use a primer but requires the presence of nucleoproteins (NP). Despite extensive genetic and biochemical studies, mechanistic details underlying the formation of active transcription and replication complexes are poorly understood. Difficulty in sample preparation has been one of the major obstacles in studying the structure of influenza virus polymerase. Using a new expression strategy, the investigator has purified, characterized and made preparative amounts of heterotrimeric influenza virus polymerase. The long-term objective of the proposed research is to understand how influenza virus polymerase, NP, the viral RNA, and other essential components coordinate their functions to ensure both cap-dependent and cap-independent RNA synthesis. The specific aims are to: (1) use cryo-EM 3-D reconstruction to elucidate the structure and dynamics of influenza virus polymerase during initiation and catalysis of RNA transcription by studying the structures of five functional intermediates; (2) determine the structure of the polymerase subunit PA and its role in RNA synthesis using biochemical assays and X-ray crystallography; and (3) elucidate the role of NP in RNA synthesis by studying (a) the effects of NP binding on the heterotrimeric polymerase structure and function and (b) the effects of RNA binding on the structure of NP oligomers.